The present invention relates to an in-situ microscope device for reactors, such as for example bioreactors.
With such in-situ microscope devices, examinations can be carried out on specimens of the material in the inside of the reactor during ongoing operation, for example the concentration of particular cells in the medium can be monitored. The basic principles of in-situ microscopy for reactors are described in patent specification DE 40 32 002 C2.
An in-situ microscope device with the features of the precharacterizing clause of claim 1 is described e.g. in the dissertation xe2x80x9cIn-situ-Mikroskopie; Ein neues Verfahren zur Online-Bestimmung der Biomasse bei Kultivierungsprozessenxe2x80x9d [In-situ microscopy: a new process for online determination of biomass in cultivation processes], Dr. Christoph Bittner, Hanover, 1994.
The monitoring and controlling of biotechnological processes has gained a major significance in the recent past e.g. in the chemical and pharmaceutical industries. Examples of this are the synthesis of human proteins, such as e.g. interleukin (IL-2), tissue plasminogen activator (t-PA) or antithrombin (AT-III), the preparation of which with the help of organic synthesis can be achieved only with difficulty, with the result that the manufacture of these proteins with the help of the cultivation of mammal cells is preferred. Microorganisms, here in particular yeasts, are used also in the manufacture of products of the food industry, e.g. beer, wine, cheese or bread. Further products or pharmaceuticals are produced by the cultivation of other organisms. In the case of in-situ microscopy, a microscope probe is inserted into a connection port of a fermenter (reactor) in order to monitor and control such processes. This microscope probe enables an image to be photographed directly in the culture stock. The photographed microscope image is photographed and digitized by a CCD camera connected to the in-situ microscope. The evaluation of the digitized microscopic images is carried out with the help of image-processing programs on a standard computer. Information about cell sizes and biomass, cell-size distribution, cell concentration, cell morphology and cell vitality can be obtained using the image data material obtained with the in-situ microscope and analyses applied to it. On the basis of the information, thereby obtained during ongoing operation, about the state of the system located in the reactor, process parameters can be influenced and controlled in order to achieve a desired development of the system.
An in-situ microscope for the observation of cultivation processes in yeasts is described in the above-mentioned dissertation by Bittner. The microscope has a dip tube which is inserted into a reactor connection port. In the end-section of the dip tube lying in the inside of the reactor, an inlet is provided through which the culture medium can flow freely. A microscope external tube is arranged coaxially in the dip tube and, with its lens lying at the inner end, is directed towards a specimen zone which is defined between the cover glass of the lens and a slide glass body lying opposite. Connected to the opposite end is the microscope external tube with a camera for photographing the image of the specimen zone. If the specimen zone is open, the lens cover glass and the slide glass body lying opposite lie at some distance from each other, the culture medium from the inside of the reactor being able to flow freely through this space. In order to photograph the image, the specimen zone is closed by moving the slide glass body onto the lens cover glass until a sealing ring surrounding the slide glass body comes to rest against the lens cover glass and thus creates a specimen zone with a defined volume between the slide glass body and the lens cover glass. In the known device, the specimen zone is closed by pulling the slide glass body with an illumination unit below it against the lens. This movement is achieved via a pull rod which runs in longitudinal direction in the dip tube alongside the microscope external tube, and is connected at one end to the unit of the slide glass body and at the other end to a drive outside the dip tube. Furthermore, a wiping apparatus is provided with which the lens cover glass is intended to be cleaned by wiping off if required. Such a wiping apparatus is necessary as the glass rapidly becomes dirty and another cleaning method cannot be carried out at all while cultivation is in progress and, even after the cultivation process is stopped, can be carried out only if considerable effort is expended and the microscope is completely removed from the reactor. The wiper is also driven by an external drive via a mechanical power transmission means.
The known in-situ microscope device is disadvantageous in various respects. For example, it is disadvantageous that mechanical power transmission means alongside the microscope tube must be guided through the dip tube, as this restricts the space available for the microscope tube. Furthermore, such mechanical power transmission means are costly in design terms and are incident-prone.
However, the main disadvantage of the known in-situ microscope devices is that the microscope is not accessible while cultivation is in progress because, if the reactor is opened by removing the dip tube or by pulling out the microscope, the cultivation would be contaminated. Furthermore, if the microscope was installed at the side, the reactor would first have to be emptied, which for practical use is out of the question.
The removability of the microscope is of significance not only for cleaning purposes during the operation, but also for the sterilization/autoclaving of the reactor system before commissioning, as temperatures of over 120xc2x0 C. are used.
For industrial-scale applications of in-situ microscopy, microscope devices are required which are robust, flexible and easy to handle.
The object of the present invention is therefore to create an in-situ microscope device, the sensitive parts of which are accessible at any time, without having to interrupt the cultivation process or endangering it through a contamination.
The characterizing features of patent claim 1 in conjunction with its precharacterizing clause serve to achieve this object. Advantageous versions of the invention are listed in the dependent claims.
According to the invention, it is provided that the dip tube is guided movable in axial direction in the reactor connection port, to which a rinsing chamber with sealable openings is externally connected, through which cleaning agents can be fed in. The dip tube can be pulled back into the connection port until the inlet of the dip tube communicates with the rinsing chamber. Sealing means are provided at the dip tube in order to keep the internal space of the reactor sealed off from the rinsing chamber when the dip tube is pulled back into the rinsing chamber. In this way, the specimen zone can be cleaned, when the dip tube is pulled back into the rinsing chamber, by feeding cleaning agents into the rinsing chamber. The internal space of the reactor remains sealed off from the inside of the rinsing chamber in order that the specimen zone can also be cleaned while cultivation is in progress, without the danger of contamination.
Furthermore, the microscope external tube can be pulled out when the dip tube is pulled back into the rinsing chamber. Thereby, all parts of the microscope device which are arranged inside the dip tube can be removed and if necessary replaced or repaired, without the sterile barrier to the inside of the reactor being broken through. Thereby, changes to the design and fitting of the microscope, maintenance work and the like can also be carried out during ongoing operation of the reactor, without the cultivation process in the reactor having to be interrupted, or the danger(of contamination. Thereby, handling during ongoing operation, operational reliability and variability can be decisively improved by replacing components of the microscope, with the result that the in-situ microscope device is particularly well-suited to industrial applications due to its flexibility and robustness.
In an advantageous version, there is accommodated in the end of the dip tube pointing towards the inside of the reactor an illumination unit which carries the slide glass body and has a light source in order to illuminate the specimen zone through the slide glass body.
In an advantageous version, the microscope external tube is for its part housed in a microscope-housing tube, wherein the microscope-housing tube is closed at the end facing the inside of the reactor except for an inlet for specimen material and surrounds the specimen zone from the rear, and wherein the illumination unit is arranged at the inward-lying end of the microscope-housing tube. The microscope external tube is advantageously housed movable in axial direction in the microscope-housing tube and a drive is provided which acts on the outward-lying end of the microscope external tube. The microscope external tube can be moved relative to the microscope-housing tube by the drive, in order thus to be able to open and close the specimen zone between the lens cover glass at the microscope external tube and the slide glass body of the illumination unit by pushing forward and pulling back the microscope external tube controlled by the drive. Alternatively, at the inner end of the microscope-housing tube a drive can be provided which acts on the illumination unit housed movable in axial direction in the microscope-housing tube, in order to be able to open and close the specimen zone by moving the illumination unit. Particularly advantageously, a step motor or a regulated direct-current motor is used as a drive, whereby a very precise setting of the specimen zone can be achieved.
In an alternative version, the illumination unit and the microscope external tube form two separate units which are not housed as above in a common microscope-housing tube, the illumination unit being arranged at the inner end of the dip tube and the microscope external tube being housed movable in axial direction directly in the dip tube, the microscope-housing tube in the version described above thus being dispensed with. Furthermore, a drive is provided which acts on the end, lying outside the reactor, of the microscope external tube in order to move this relative to the dip tube in a controlled manner, in order thus to be able to open and close the specimen zone between the lens cover glass at the microscope external tube and the slide glass body of the illumination unit by pushing forward and pulling back the microscope external tube.
In all versions, it can be provided that a microscope tube is housed movable in the microscope external tube and that a drive means is provided in order to be able to move the microscope tube in longitudinal direction in a controlled manner, in order thus to be able to set the lens against the microscope tube relative to the specimen zone for focusing.
With the previously described forms, it is possible to exploit the cross-section available in the dip tube as far as possible, because no drive transmissions need to be guided through the dip tube. Furthermore, the position of the slide glass body and the lens cover glass relative to each other, which between them form the specimen zone, can be set very accurately in a controlled manner by the drive.
The fact that the cross-section area of the dip tube can be fully exploited, because no drive transmissions need to be guided through, means that dip tubes with a relatively small internal diameter, into which a microscope external tube is introduced, can also be used. In this way, dip tubes of conventional, standardized exchangeable probe systems, in which the external diameter of the probe to be used is limited, can also be used.
If the dip tube is pulled back into the rinsing chamber, cleaning agents, e.g. superheated steam, can be fed in through the sealable openings of the rinsing chamber in order that in this way the cleaning agents enter the specimen zone through the inlet of the dip tube, in order to clean the specimen zone. If the dip tube is pulled back until its inlet communicates with the rinsing chamber, the inside of the reactor is sealed off and the inlet of the dip tube lies in the inside of the rinsing chamber and no longer communicates with the inside of the reactor. In this situation, the microscope external tube or the microscope-housing tube can be pulled out of the dip tube. All essential parts of the microscope are thereby accessible and can be repaired or varied by replacing components. The last-mentioned possibility of changing the properties of the microscope by replacing components also applies to the specimen zone, because e.g. the slide glass body can be replaced in order to obtain another geometric definition of the specimen zone.
If a drive is provided in order to set the specimen zone by moving the microscope external tube or the illumination unit relative to each other, an accurate definition of the specimen zone is possible through such a precisely controllable drive. The motor control allows a variable configuration of the specimen zone, i.e. the height of the specimen zone to be selected through variable approaching. A supplementary or alternative option of a variable configuration of the specimen zone is offered by particular forms of the slide glass body which are described in the following.
In this regard, it is preferred in particular that the slide glass body has a sapphire glass plate with a level external area, on the circumference of which an annular rim of a predefined thickness is formed which serves as a spacer if the lens cover glass is moved against the slide glass body until it rests against the annular rim in order to close the specimen zone. By keeping ready slide glass bodies with annular rims of various thicknesses, the height of the specimen zone can be varied by using a selected slide glass body. The annular rim can be formed e.g. by the polishing in of a recess into a sapphire glass plate or produced by applying an annular material layer.
Instead of an annular spacer, several discrete spacer bodies can also be formed on the sapphire glass plate, e.g. two oblong spacers which between them form a channel-shaped recess open at both sides. In versions in which several discrete spacers are provided at a distance from each other on the slide glass body, the specimen zone is open at the side opposite the surrounding medium in order that cells can flow continuously through the specimen zone and therefore the image recorded by the microscope continuously changes. A higher measuring frequency can thus be achieved by the photographing of image sequences because the specimen zone need not be opened and closed for each image, rather a complete replacement of the medium in the specimen zone should be effected by the opening of this only at certain intervals. This version of the specimen zone differs significantly from the specimen zones closed on all sides, which serve exclusively to keep the specimen steady in the specimen zone. The opposite is the case in the variant proposed here. When using LEDs of high light intensity for illumination, the exposure time of the camera can be shortened such that the cells are represented sharply despite their movement.
The in-situ microscope device according to the invention can operate with a finite or an infinite lens.
Furthermore, in addition to the already-described operating method of transillumination bright-field microscopy, the in-situ microscope device can also be used with an illumination unit below the specimen zone for direct-light darkfield microscopy or for direct-light bright-field microscopy.